In Vitro Expression And Functional Characterization Of Three Laodelphax Striatellus P450s On Imidacloprid Metabolization

The small brown planthopper（SBPH）,Laodelphax striatellus（Fallen）is an important cereal pest in China.The application of insecticide is the main measure to control the SPBH,and with the large amount of insecticide application,the SPBH has developed resistance to various commonly used insecticides,such as imidacloprid and chlorpyrifos.Studies on the resistance mechanism of the SPBH showed that the enhancement of cytochrome P450 enzyme activity is the main reason for the resistance of the SPBH to imidacloprid.Research confirmed that the over-expressed CYP6AY3v2 and CYP353D1v2 enzymes in Imidacloprid-resistant strains of SPBH have metabolic effects on imidacloprid.Due to the evolutionary plasticity of cytochrome P450 enzymes,there may be other P450 enzymes in SPBH that have potential metabolic effects on imidacloprid.In order to identify these P450 enzymes,in the early stage of this study,using transgenic technology,three enzymes CYP6CW1,CYP6FK1 and CYP380c10 were screened from 63 P450 genes of SPBH,which may have metabolic effects on imidacloprid.In order to verify the screening results,three P450 enzymes,CYP6CW1,CYP6FK1 and CYP380c10,were expressed in vitro,the enzyme activity was measured and the metabolism of Imidacloprid in vitro was studied.The results are summarized as follows:1.In vitro expression of three P450 enzymes of the SPBHUsing the insect baculovirus expression system,the above three P450 genes and CPR were expressed in vitro.The plaque analysis method measured the storage titer of P3 generation virus with no load of FastBac-HTA,CPR and three P450 enzymes,respectively 7.0 × 107（pfu/mL）,6.0 × 107（pfu/mL）,4.0× 107（Pfu/mL）and 5.0 × 107（pfu/mL）.Three P450 enzymes P3 generation viruses and CPR were added to High Five cells with MOI values of 2 and 0.2 respectively for in vitro expression.The protein content and spectral characteristics of recombinant CYP6CW1,CYP6FK1 and CYP380c10 were determined by CO difference spectroscopy.T he results show that these three recombinaned P450 enzymes have obvious absorption peaks at 450 nm after they are combined with CO,indicating that the three P450 proteins are stable in nature and have the characteristics of P450 enzymes;Secondly,the recombinaned CYP6CW1,CYP6FK1 and CYP380c10 enzyme contents are 284.25±4.12pmol/mg protein,138.42±2.59 pmol/mg protein and 311.09±4.64 pmol/mg protein,respectively.2.Determination of the metabolic activity of four model substrates by three P450 recombinaned enzymesThe metabolic activities of four substrates,namely,p-nitroanisole（PNA）,7-ethoxycoumarin（EC）,7-benzyl-4-trifluoromethylcoumarin（BFC）and 7-methyltetrazoline（MR）,were determined.The results showed that:compared with the control group,the recombinant expressed CYP6CW1,CYP6FK1 and CYP380c10 showed higher activity of oxygen deacetylation for EC,and the activity ratio was between 2.16 and 3.56;CYP6CW1 and CYP6FK1 showed higher oxygen demethylation activity to PNA,with specific activity values of 3.36 and 2.59,respectively;Only CYP6CW1 showed oxygen demethylation activity on MR,and the activity ratio was 2.23;All three enzymes had no oxygen debenzylation activity to BFC;The above results show that the three recombinaned expressed P450 enzymes show different activities on different substrates.3.In vitro metabolism of imidacloprid by three P450 recombinaned enzymesRecombinant P450 CYP6CW1,CYP6FK1 and CYP380c10 were separately incubated with imidacloprid.The samples without NADPH regeneration system and empty HTA plus NADPH were used as negative controls.The production rate of 5’-hydroxyimidacloprid was detected by UPLC-QTOF MS with ultra-high performance liquid phase-quadrupole time-of-flight mass spectrometry,The metabolic activity of P450 enzyme to imidacloprid was expressed by the formation rate of 5’-hydroxyimidacloprid.The results showed that CYP6CW1,CYP6FK1 and CYP380c10 were detected with 5’-hydroxyimidacloprid after incubation with imidacloprid,the production rate were 0.0198±0.0008 pmol product/min/pmol P450,0.0172 ± 0.0005pmol product/min/pmol P450 and 0.0055±0.0001pmol product/min/pmol P450,respectively.The results of further enzyme kinetic study showed that the production of 5 ’hydroxy imidacloprid was time-dependent when the recombinant CYP6CW1,CYP6FK1 and CYP380c10 metabolized imidacloprid;The kinetic parameters of CYP6CW1,CYP6FK1 and CYP380c10 were also measured.The Km values were 79.86±6.46 μM,58.69±2.61 μM and 67.15±3.24 μM,respectively.Furthermore,the intrinsic clearance rates（Vmax/Km）of the enzyme metabolic efficiency were obtained as follows 0.00025,0.00029 and 0.00008.In conclusion,the results of this study confirmed the effect of CYP6CW1,CYP6FK1 and CYP380c10 on the metabolism of imidacloprid,and provided evidence for the possible involvement of CYP6CW1,CYP6FK1 and CYP380c10 in the formation of imidacloprid resistance.